In spite of clear functional importance of CD28 in T cell activation, we still do not have a complete understanding of how CD28 transduces proximal signals. CD28 costimulation has been associated with a variety of downstream signaling pathways, but most of these overlap with TCR pathways initiated by TCR and CD28 is often thought of as an amplifier of TCR signaling. Thus, it has been difficult to assign a specific function to CD28. Most of the focus has been on the short cytosolic tail domain (CTD) of CD28 that contains two well established protein binding motifs. However, mutation of these motifs in the endogenous CD28 locus have only partial effects on T cell activation, even when both sites are mutated together. These data indicate that additional signaling motifs/pathways are required to account for the full effect of CD28 costimulation. Therefore, we are proposing an unbiased proteomic approach to identify any proteins that are selectively colocalized with CD28 during T cell costimulation. This approach does not depend on direct or stable interaction with CD28 and has the potential to identify novel proteins that have not previously been implicated in CD28 costimulation. We will achieve this goal through two Specific Aims: 1. Identify the proteome colocalized with CD28 during T cell costimulation. A major contribution to the magnitude, specificity, and regulation of cell signaling is mediated through colocalization of receptors with proximal and downstream signaling components. Proximity labeling is a relatively new technique that has been developed to identify critical colocalization events that are not detectable by conventional biochemical approaches. The use of APEX2 affords both a spatial and temporal resolution that allows for the identification of proteins that are colocalized with the target protein, without requiring stable physical association. In this Aim we will apply this technology in combination with SILAC (Stable Isotope Labeling by Amino acids in Cell culture), LS-MS/MS to quantify and sequence peptides, and established search programs and protein databases to identify proteins that are colocalized with CD28 during T cell activation and costimulation. 2. Determine the role of colocalized proteins in CD28 costimulation. In this Aim we will determine the relative contribution of candidate proteins that we have found to be colocalized with CD28 during costimulation through a combination of biochemical, molecular, cellular and immunological assays. 1